1,25-Dihydroxyvitamin D3 activates Raf kinase and Raf perinuclear translocation via a protein kinase C-dependent pathway.

Abstract

1,25-Dihydroxyvitamin D3's (D3) potential mitogenic mechanism of action was pursued in cultured rat hepatic Ito cells, a fibrogenic effector cell which proliferates in vivo during liver injury and fibrogenesis. D3 stimulated Ito cell DNA synthesis and potentiated platelet-derived growth factor-induced mitogenesis. D3's enhancement of [3H]thymidine incorporation was associated with nuclear Egr expression. Recent studies have causally linked the activated proto-oncogene c-Raf with downstream Egr induction. The serine-threonine kinase Raf protein is phosphorylation-activated by a large array of agonists including plasma membrane and cytoplasmic tyrosine kinases but has not previously been associated with the steroid superfamily of mediators. To consider potential prenuclear acute pathways of D3-induced stimulation, the activation of Raf was examined following D3 exposure. D3 induced Raf activation as assessed via (a) enhanced Raf phosphorylation following in vivo 32P labeling, (b) enhanced kinase function utilizing exogenous histone 1 protein as substrate, and (c) the shift in Raf physical localization changing from a diffuse cytoplasmic distribution to a perinuclear domain. A similar activation of Raf kinase was found in 3T3 cells exposed to D3 with enhanced histone phosphorylation detectable within 1 min following stimulation. The proximal cascade leading to Raf kinase activation may involve a protein kinase activity was severely attenuated by stimulated kinase activity was severely attenuated by previous phorbol ester treatment for 20 h or staurosporine pretreatment.